Project Summary/Abstract Mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 confer an increased lifetime risk of breast and ovarian cancers. Clinically, BRCA-deficient tumors are sensitive to platinum-based chemotherapeutics and poly-ADP ribose polymerase inhibitors (PARPi). However, resistance to PARPi presents a challenge for effective BRCA-deficient cancer treatment. In addition to their established roles in homologous recombination, BRCA proteins play an emerging role in protecting replication forks from extensive nucleolytic degradation. Stalled or damaged replication forks reverse their course to aid in the repair of DNA damage, and these reversed replication forks are the substrates for extensive degradation by nucleases. Notably, extensive nucleolytic degradation of reversed replication forks is not a terminal event because BRCA-deficient cells activate recovery mechanisms to cope with extensive degradation. The overall goal of this project is to determine the mechanism of replication fork recovery in BRCA1-deficient cancer cells, which will contribute to development of novel chemotherapeutic strategies for BRCA-deficient tumors. My preliminary data implicate the Rad18 protein in the fork recovery mechanism of BRCA1-deficient cancer cells. Moreover, I found that loss of Rad18 in BRCA1-deficient cancer cells exacerbates replication fork degradation. Rad18 monoubiquitinates proliferating cellular nuclear antigen (PCNA), promoting recruitment of Translesion Synthesis (TLS) polymerases to cope with DNA damage. On the basis of my preliminary data and Rad18?s established ubiquitination activity, I hypothesize that PCNA monoubiquitination modulates replication fork recovery and plays a novel role in fork protection in BRCA1- deficient cancer cells. Aim 1 of this proposal will test whether BRCA1-deficient cells activate a Rad18- and PCNA monoubiquitination-dependent mechanism of replication fork recovery. Furthermore, this aim will determine whether this mechanism requires specific TLS polymerases to recover the stalled forks. Aim 2 will define how Rad18 and/or PCNA monoubiquitination mediate replication fork protection. I will use a unique combination of single-molecule DNA fiber assay and electron microscopy approaches to accomplish Aims 1 and 2. Aim 3 will test the clinical relevance of exploiting the Rad18 and PCNA ubiquitination pathways therapeutically in BRCA1- deficient cancers. I will utilize both cultured cell lines and tissue microarrays to test whether Rad18, TLS polymerases, or PCNA ubiquitination can be effectively targeted to modulate chemoresponse in a BRCA1- deficient background. The outlined experiments will contribute to novel therapy development, with the ultimate goal of combating growing chemoresistance in BRCA-deficient tumors.